Biosynthetic mRNA capture of whole-genome thiol-labeled and nonlabeled mRNAs during asexual and sexual development. (A) Expression of FCU-GFP from the cam promoter occurs in the parasite strains 3D7 and F12 regardless of developmental stage. 3D7 produces gametocytes, and the transcriptional dynamics in 3D7cam are representative of both asexual (A) and committed gametocytes (cG). Expression of FCU-GFP from the pfs16 promoter in 3D7pfs16 captures mRNA dynamics only in parasites committed to gametocytogenesis (cG). F12 is not able to produce mature gametocytes, and mRNA dynamics occurring prior to gametocytogenesis can be measured regardless of the promoter used. (B) Thiol-tagged RNA was separated from the total pool by streptavidin magnetic purification. mRNAs eluted from the beads are 4-TU labeled (representing “transcription”); (C) unbound mRNAs were present before the addition of 4-TU (representing “stabilization”). Each column represents the Log2(Cy3/Cy5) ratio for every gene in the sample detected above background for mRNAs transcribed (5168 genes) or stabilized (5175 genes) at 0, 12, 24, or 36 h post-invasion (hpi). Ratios for each gene were K10 means clustered (1–10) and ordered according to their peak value starting at 0 hpi. The median Pearson's r for each gene over time between strains is shown below the respective time-course. The fold change in transcription and stabilization for each gene (Log2[Cy3/Cy5] ratio of 3D7pfs16/3D7cam and F12pfs16/F12cam) was calculated and represented to aid in determining enrichment in the gametocyte-specific population. (*) Clusters highlighted in the text.
